The goal of this project is to understand innate immune responses to mycobacteria. Mycobacterium tuberculosis is responsible for more human deaths than any other bacterial pathogen. It is unique among bacterial pathogens in that it is able to subvert host defenses and establish persistent, latent infection. When host immunity wanes, the mycobacteria can reactivate and cause disease. There is little molecular understanding of bacterial pathogenesis or host responses. Effective host responses likely depend upon recognition of mycobacterial patterns and activation of innate immune responses. Macrophages serve as the site for mycobacterial replication but also are central to host defense. We hypothesize that there are host factors important in killing mycobacteria, as well as bacterial factors that enable the pathogen to subvert destruction. We propose functional genomic and genetic approaches to identify and characterize innate immune responses to mycobacteria. Using Drosophila phagocytic cell lines, we will develop an in vitro cell based assay to study mycobacterial infection of macrophages. We propose a genome-wide analysis, using the cell-based assay and an RNA interference (RNAi)-based approach, to identify host factors important in mycobacterial entry into and survival within phagocytes. Secondary assays will be done to characterize the role of the host factors during infection and their relationship to bacterial virulence factors. Microarray experiments will be done to characterize signaling pathways that are activated in Drosophila macrophages in response to mycobacteria. We will examine the host factors that we identify in RNAi screens and microarray analysis for their role in mycobacterial infections in flies and in mammalian macrophages. This project, carried out in the laboratories of Dr. Norbert Perrimon at Harvard Medical School and Dr. Eric Rubin at the Harvard School of Public Health, will enable the candidate to establish an independent laboratory studying pathogenesis of M. tuberculosis infection with a focus on host innate immune responses.